1
Q
What does proteostasis ensure
A
correct folding, concentration and location
2
Q
Aspects of proteostasis
A
- protein synthesis
- protein QC maintenance
- protein degradation
3
Q
Protein synthesis
A
- Co or post translational folding
- correct concentration
4
Q
Protein QC maintenance
A
- unfolding and refolding
- correct location
5
Q
Protein degradation
A
- correct concentration
- destroy aggregates
6
Q
Cellular stressors
A
- temperature
- chemical
- oxidative
- osmotic
- mutations
7
Q
How does heat affect protein folding
A
breaking of VDW and H bonds leading to denaturation but can also lead to bond reformation
8
Q
2 types of proteins
A
- stable
- unstable
9
Q
stable proteins
A
- common
- low propensity to aggregate
- less sensitive to stressors
10
Q
unstable proteins
A
- have intrinsically disordered regions for flexibility
- sensitive to misfolding
- sensitive to temperature due to loss of a small set of crucial effectors and regulators of biological processes
- biosensor for stress
11
Q
Concentration affecting protein folding
A
- correct folding is impaired by high protein concentration due to overloading chaperones so aggregates occur
- possible reason for trisomy 21 leading to increased alzheimers as APP gene is found on chr21
12
Q
How is protein misfolding toxic
A
- loss of normal biological function
- gain of toxic functionG
13
Q
Gain of toxic function
A
- clog up intracellular transport/degradation
- induce inflammation
- sequester other proteins
14
Q
How do cells prevent/cure misfolded proteins
A
- halt transcription/translation
- degradation
- increase chaperones for folding and refolding
- cell death
15
Q
When do chaperones function
A
- during synthesis = prevent peptide folding before domain is complete
- during folding = help partially folded intermediates to cross energy barriers
- during misfolding = unfold and refold proteins
16
Q
Intracellular protein clearance pathways
A
- proteasome
- autophagy
17
Q
The discovery of the heat shock response
A
- found in drosophila salivary gland chromosomes
- observed puffing induced by heat shock or chemical stressors
- puffing was rapid, reversible and positively stains for RNA
- heat shock caused a change in the types of protein expressed thus RNA
18
Q
heat shock response
A
- induced at 15-15 degrees above optimum growth temperature
- increased transcription/translation of heat shock proteins
- aids survival of stimulating stress
- primes for survival of subsequent stress
19
Q
how does priming by the heatshock response help survival
A
- switches cells to making heat shock proteins
- prepares cells to combat following stress
20
Q
Hit5 seedlings - heat shock response
A
- mutant Hit5 seedlings can survive a 44 degrees heat shock without priming
- mutation increases basal levels of heat shock protein mRNA
21
Q
Transciptional changes allowing HSPs to become a major protein product
A
- repress transcription of most mRNAs
- increased transcription of specific mRNAs
22
Q
What does the duration of gene expression changes correlate with
A
severity of stress and may also be stress specific
23
Q
Repression of transcription via SINES
A
- act like transcription factors
- transcribed by RNA Pol III during stress
- bind and inhibit RNA Pol II transcription
- when SINE RNA binds RNA Pol II it keeps the pre-initiation complex (PIC) in the closed conformation
- PIC cannot access DNA for transcription
24
Q
Increased transcription via HSF1
A
- sensor of protein misfolding which is normally kept as a monomer but in some species it does have basal activity
- HSF1 is a monomer which is bound to Hsp70,40,90
- stress causes Hsp90 to bind misfolded proteins thus will unbind HSF1
- HSF1 can now form a homotrimer
- homotrimer binds to promoters containing heat shock transcription elements in heat responsive target genes
- binding to promoters allows RNA pol II to dissociate from NELF and DSIF
- HSF1 recruits P-TEFB to phosphorylate promoters causing NELF to be released
- RNA Pol II is now free and can transcribe heat shock proteins
25
HSPs chaperone protein folding mechanism
- open position with ATP bound
- misfolded protein is loaded
- ATP to ADP causing lid to close
- closing of lid protects hydrophobic components allowing for remodelling
- ADP to ATP
- protein released
- can be constitutive or inducible
26
Stress granule assembly order
cores then shell
27
Stress granule disassembly order
shell then core
28
3 steps of stress recovery
- dephosphorylation of pEIF2a
- SG disassembly
- resumption of translation
29
Dephosphorylation of pEIF2a
- depletion of ternary complex leads to the translation of ATF4
- ATF4 helps chaperones to transcribe and translate CHOP
- CHOP induces Gadd34
- Gadd34/CreP binds PP1 which inhibits phosphorylation of EIF2a
30
SG disassembly
- mRNAs are released to polysomes for translation
- done through surface exchange of loosely interacting proteins on shell with polysomes and remodelling via disaggregase chaperones
31
SG disassembly - disaggregase chaperones
- AAA ATPases
- homohexamer of 6 B6P monomers
- central hydrophobic channel for proteins to thread through
- unfolds UB substrates and unpicks protein complexes in the presence of ATP
- KO leads to abnormal presence of stress granules
32
Resumption of translation
- increase in ternary complexes
- polysome formation
33
ALS mutations in SG proteins
- can recruit more SG proteins to SG
- decreased recovery when TIA1 mutants
34
CHOP as a transcriptional activator and repressor
indices DDIT3 which activates pro-apoptotic genes and represses anti-apoptotic genes
35
Pro-apoptotic genes
- trail receptor (extrinsic)
- Bim (intrinsic)
36
Anti-apoptotic genes
- Bcl-2
- Bcl-XL
37
Why do cells die
- CHOP induced
- defence
- development
- homeostasis
38
How is cell death classified
- morphology
- biochemistry
39
Apoptosis morphology
- cells shrink
- organelles remain intact
- condensed chromatin
- controlled DNA fragmentation = ladders
- apoptotic bodies engulfed by phagocytes
- cell blebbing
40
Necrosis morphology
- cell and organelle swelling
- moderate chromatin condensation
- random DNA fragmentation = smear
- cell lysis
- inflammatory products produced
41
Autophagic morphology
- accumulatio of double membraned vacuoles
- lack of chromatin condensation
- little or no phagocytotic uptake
42
Accidental cell death (biochem classification)
- cell murder
- always necrotic (not all necrosis is ACD)
- harsh injury that cell cannot recover from
- no adaptive response and no signalling regulation
- release of inflammatory molecules = DAMPs/alarmins
- DAMPs/alarmins then activate regulated cell death in nearby cells
43
Regulated cell death (biochem classification)
- cell suicide
- can be not stress driven (development) or stress driven
- defined signalling pathways
- morphology is apoptotic or necrotic
- stress driven ways to die = apoptosis, necroptosis, autophagy
44
Extrinsic apoptosis
- Ligand causes receptor (fas etc) to form a homotrimer
- formation of homotrimer brings together the FAS associated death domains
- leads to the recruitment of procaspase 8 which is cleaved leading to active caspase 8
- caspase 8 cleaves procaspase 3 and Bid
- formation of tBid can upregulate pro-apoptotic genes (link to intrinsic)
- Caspase 3 dismantles cell via cleaving cytoskeleton and DNA = apoptotic body forms
45
Intrinsic apoptosis
- not activated by an extracellular ligand but instead cell senses stress
- increased pro-apoptotic genes and decrease in anti-apoptotic genes via BH3 only proteins activating Bax/Bak
- pro-apoptotic molecules can homo oligomerise to form a pore/channel in the mitochondrial OM
- release of cyt c, APAF1 and procaspase 9 to form a apoptosome
- apoptosome cleaves procaspase 3 = apoptosis
46
What is necroptosis
an alternative mode of RCD mimicking features of apoptosis and necrosis
47
When is necroptosis common
when caspase 8 is inhibited and FADD is depleted as the apoptotic pathway cannot be activated
48
How is necroptosis activated
same way as extrinisic apoptosis
49
What does necroptosis require
death domain containing kinases such as RIPK3
50
Necroptosis morphology
same as necrosis
51
RIPK1 as a checkpoint
- can activate all 3 pathways = apoptosis, necroptosis, survival
- activated by TNF
52
RIPK1 activating cell survival - acting as a scaffold
- phosphorylation at Ser25 which is promoted by Ub of RIPK1
- acts as a scaffold where it recruits molecules required for cell survival
53
RIPK1 in cell death - kinase
- dephosphorylated at Ser25
- if caspase 8 and FADD are present then will go to apoptosis
- if both are absent then RIPK3 will be activated for necroptosis
54
RIPK3 activation by
- Death domain receptors (FAS) via RIPK1
- Toll-like receptors via TICAM1
- viral nucleic acids via DAI
- adhesion receptors
55
MLKL conformational changes
- MLKL phosphorylated by RIPK3 at its C terminus
- extension of MLKL 4 helix barrel
- conformational change allows IP6 to bind (-)
- increased affinity for membrane (+)
56
MLKL possible mechanisms in membrane
- activates TRPM7 or Na channels = influx = depolarisation = water = bursting
- permeabilises the membrane
- pore creation for ion transport = ion in = water = bursting
57
3 assays for cell death
- TUNEL
- Live/dead
- apoptotic/dead
58
TUNEL assay
- tissue or fixed cells
- imaging
- enzymatic addition of labelled dUTP which labels a 3'OH
- Detects DNA breaks formed from DNA fragmentation
- cannot distinguish between necrosis or apoptosis as both involve DNA fragmentation
- may also detect basal DSDNA breaks and cell turnover
59
Live/Dead assay
- cell cultures
- imaging or flow
- Uses 2 fluorophores
- Live = Calcein-AM cleaved by enzymes in live cells
- Dead = Ethidium homodimer/propidium iodine
- discriminates between necrotic or late apoptotic but would also show necroptosis
60
Apoptotic/dead assay
- cell cultures
- imaging or flow
- Uses 2 fluorophores
- Live = Annexin V binds to phosphatidylserine
- Dead = propidium iodine
- discriminates between necrotic or late apoptotic
- doesnt consider necroptosis
61
Cell death in cancer
- have dysregulated cell divison/death
- therapies attempt to decrease cell division or induce cell death
62
Primary cell cultures
- derived from normal animal tissue
- limited ability to proliferate
63
Transformed cells
- can be derived from cancer tissues
- differentiated primary cells transformed via oncogenic viruses
- immortal and divide continuously
64
Optimum culture growth conditions
- 37 degrees
- 5% CO2
- normoxia or hypoxia
- humidified
- growth media
- serum
- additives such as growth factors
65
Growth media components
- salts
- glucose
- vitamins
- amino acids
- buffer
66
Serum
- produced from the blood of a fetal animal
- low antibodies
- contains many growth factors
67
Factors influencing a cells fate and identity
- specified changes in gene and protein expression leading to a fully differentiated cell from embryonic stem cells
- intrinsic factors = genetic program
- environmental factors = growth media
68
Methods to determine cell identity
- morphology
- protein expression
- gene expression
69
Methods to assess morphology
- bright field = stained
- phase contrast = live cells
- fluorescence
- SEM
- TEM
70
Methods to assess protein expression
- immunohistochemical analysis
- microscopy
- flow cytometry
71
Single cell RNA sequencing for gene expression analysis
- isolates cells from tissue
- add barcoded beads
- suspended single cells
- amplify and sequence
- identifies the type and number of mRNA in each cell
- can lead to the identification of new and rare cell populations
72
Human cell atlas uses
- look at development = retina
- evolution = primate species brain dev
- find rare and new cell types = trachea
- study disease states = covid-19
- validate disease models = breast cancer
- aging = skeletal muscle changes with age
73
Somatic cell nuclear transfer
- e-nucleate egg
- add nucleus of terminally differentiated cells
- differentiate to inner cell mass stage
- isolate ESC
- add ESC to a tetraploid blastocyst where it contributes to developing embryo
- embryo develops and clone is formed
74
uses of cell fate manipulation
- growing cell types which arent accessible
- regenerative medicine
-study aging cells
75
methods to make iPSC
- transfection using DNA, RNA or protein
- viral vectors
- gene editing
- chemical induction
76
considerations when selecting a viral vector
- tropism
- integration
- lytic
- size
77
Yamanaka and Takahashi factors discovery
- used a dispensable gene = Fbx15
- inserted reporter Bgal-NeoR into Fbx15 locus
- transduced MEF with pluripotency factors
- selected for NeoR to find pluripotent cells
- found 4 factors = Oct4, Klf4, Sox2 and c-myc
- problem where it didnt contribute to developing embryo so redid experiment with nanog instead of Fbx15
- inserted GFP-puromycin-resistance cassette into 5'UTR of Nanog = generated chimaeric mice
78
How to confirm pluripotency
- morphology
- cell surface markers
- differentiation into all germ layers
- could terminally differentiate
- gene expression of factors
79
Similarities between iPSC and ESC
- morphology
- gene expression
- protein expression
- proliferative rate
- telomerase activity
- epigenome
- chimeric mice
- germline transmission
80
Differences between iPSC from ESC
- iPSC has integration of RV
- iPSC has some epigenomic memory retained from starting cell type
81
iPSC applications
- modelling aging diseases such as alzheimers (inserting mutations for familial and sporadic)
- modeling complex orders such as bipolar
- potential to make germ cells which can generate offspring
82
General steps for human iPSC based disease modeling
- extract somatic cells from patient
- reprogram cells into iPSCs
- create an isogenic control with Cripsr-Cas9
- differentiate into desired cell types
- characterise disease phenotypes and identify molecular mechanisms
83
iPSC derived cardiomyocytes - in vitro example
- hard to obtain primary cardiomyocytes so we can use iPSC to help
- human peripheral blood mononuclear cells were reprogrammed into iPSCs via OKSM episomal factors
- iPSCs differentiated into cardiomyocytes via extrinsic factors
- infected them with SARS-COV-2
- saw that it could enter and replicate via ACE2
- saw cells stopped beating in 3 days and apoptosis was induced
- developed an ACE2 antibody to treat these cells
84
Reversing aging for heart repair example - in vivo
- transient OKSM reprogramming in mice
- OKSM in genome was under a doxycycline inducible cardiac specific promoter
- restricted oxygen to mimic a heart attach
- saw rejuvination where reprogrammed cells differentiated into immature cardiomyocytes which could proliferate and repair the heart
- detected via cell surface markers
- very specific on/off switch with factors as could lead to tumour growth and death if left on too long
85
Understanding Alzheimers disease using iPSC - in vitro
- RV-OKSM hiPSC with a PSEN-1 mutation
- no normal control but had iPSCs from a patient with sporadic AD
- differentiated into cortical neurones
- mutant secreted a Tau peptide (eTau) into the media
- eTau media was then put on healthy cells and saw an increase in AB42, decreased a-secretase APP cleavage and soluble APPa
- also caused neuronal hyperactivity
86
AD drug screening using iPSC
- RV OKSM iPSC from renal tubular cells
- differentiated into normal cortical neurones
- exposed to AB42 and screened for neuroprotective agents
- found that CDK inhibitors appeared protective
87
Methods to avoid using RV
- non-integrating vectors
- chemical reprogramming
88
Non-integrating vectors
- RNA is modified to be stable and non-immunogenic
- inducible expression
- ex = episomal vectors
- alternative to RV which may lead to cancers and mutations in cells
89
Chemical reprogramming
- uses no viral vectors
- use of small molecules instead of OKSM RV
- low efficiency to protocol needs to be improved
- found via scRNA-seq that cells take a different gene expression route with this reprogramming
90
Direct reprogramming
- avoids using oncogenes which promote tumour growth when transplanted
- goes straight from differentiated cell to differentiated cell = no pluripotent cells
91
Benefits of direct programming
- faster
- more efficient
- less costly
- doesnt rejuvinate cells = good for age-related disorders
- avoids tumour growth
92
Neuron marker
TUJ1
93
Problems with 2D cultures
- can be hard to get cells to stick on plastic
- feeder layers of other cells may be needed to secrete supportive factors
- secrete GF and ECM components - not ideal when looking to transplant
- doesnt fully represent the in vivo environment
94
Benefits of 3D in vitro cultures (organoids)
- cells organise themselves into complex structures of multiple cell types
- can themselves secrete growth and supporting factors so dont need a feeder layer
- can form mini organs
- more physiologically relevant than 2D cell culture is
95
Potential organoid uses
- study development (Avoid 14 day rule)
- study disease
- evolution
- personalised drug screening
- transplants
96
Organoid production
- starting cell types = ESC,ASC and iPSC
- need a defined media of chemical factors
- series of steps that reflect the cells signalling environment within a developing embryo
- initiate and maintain normal temporal gene expression to produce correct cell identity
- often need an ECM component
97
2 types of brain organoids
- cerebral (Lancaster protocol)
- cortical (velasco protocol)
98
Lancaster protocol
- Cerebral organoids
- self organised from hESC or LV-OKSM iPSC
- differentiate themselves in culture = follow own developmental profile
- have forebrain, midbrain and hindbrain
- cells plated in a suspension media
- neural induction media
- differentiation media and matrigel
- spinning bioreactor
- problem = each brain produced is different
99
Velasco protocol
- Cortical organoids
- defined media with chemical signals to direct differentiation into cortex
- little variation
100
Multi cell organoids
good for when studying physiology and for organ replacement
101
Single cell organoids use
- drug efficacy and toxicology
- genetics
102
Organoid limitations
- size
- lack of vasculature = lack of nutrients
- not complex enough, cant study aging
- variable
- ethical concerns
103
Gut assembloid
- Tabeke et al 2021 took iPSC derived gut spheroids differentiated into anterior and posterior
- fused these spheroids together in matrigel
- spheroids signalled to one another leading to the development of a pancreas, liver and bile duct
- allowed for the modelling of a gut disease caused by HES1 mutant
- growth was confirmed via a PROX1-tdTomato reporter system
104
Assembloids
3D structures that incorporate 2 or more organoids/spheroids that are fused together to form a multi regional or multi-lineage assembloid
105
Brain assembloid
- Sergiu P. Pascas group
- fused a cortical organoid with a striatal organoid
- also fused a cortical-spiral-cord-muscle assmbloid
- saw that stimulation of neurones triggered muscle contraction
106
Somatosensory assembloid
- 4 part organoid
- combination of cortical, diencephalic, spinal and sensory
- sensory pathways
- responsive to chemical signals
- used it to model SCN9A mutations which lead to no pain = due to abnormal signalling and inefficient signals reaching the brain
107
Organ on a chip models
- allows for vasculature
- flow system with cells which will form endothelial networks
- blood vessels organoids form throughout organoids allow for increased growth, maturation and function
108
Venom secretory organoid
- solves problem of the difficulting in developing antivenoms
- AdSc derived
- venom could be harvest from media
109
Problems with venom secretory organoids
- different development
- cold blooded animals so have to be cultured at a different temperature
- growth media formulation
110
SARS-COV2 organoid modelling
- Lancaster developed a choroid plexus organoid which secretes CSF
- covid infected and broke down choroid plexus cells allowing for CSF fluid containing SARS and inflammatory molecules to pass the blood-brain barrier
- intestinal organoid found that SARS can be passed through fecal matter
- proved Hydroxychloroquine didnt target endocytosis of virus = not a good treatment
111
Zika virus infection mechanism using organoids
- cannot be modelled in a mouse due to their different brains
- cortical organoids generated and used to determine pathology
- found it preferentially effected neural progenitor cells
- screen for drugs
112
Autism modelling with organoids
- CHD8 is a common mutant in ASD
- mutant iPSC generated using CRISPR from human skin and differentiated into cerebral organoids
- used a isogenic control
- saw alterations in inhibitory neuronal differentiation
113
Ex vivo
cells cultured in the lab before transplant back into patient
114
Autologous cell therapy
cells used are derived from the patient
115
Autologous cell therapy advantage
wont be rejected so no immunosuppression needed
116
Allogenic cell therapy
human origin but from an individual distinct from patient (cell banks)
117
Allogenic disadvantages
will need to be on immunosuppressants
118
Allogenic advantages
- good if patient has lost cells
- decreased culture process required
119
Xenogenic cell therapy
non human origin
120
Xenogenic benefit
more accessible and cheaper
121
In vivo
cells modified within a living animal or human
122
Good manufacturing process
- a set or principles and procedures that when followed helps ensure that therapeutic goods are of high quality, safe and potent
- regulations differ by country
123
Quality control for cells
- no contaminants
- cells produced are the same every time
- contains no pluripotent cells in order to avoid tumors
- sterility
- use of animal free media
124
3 different CRT to cure blindness
- ASC
- iPSC derived ocular organoid
- autologous iPSC derived retinal pigment epithelial cells
125
ASC blindness CRT
- autologous
- culture of adult corneal stem cells extacted via biopsy
- grown into a sheet of cells on a sample of amniotic membrane
- grown on a MEF feeder layer = possible contamination
126
iPSC derived ocular organoid to treat blindness
- self patterened
- mimics whole eye development
- corneal epithelial zone cells grown into a sheet and transplanted into mice showed recovery
- some recovery in human trials
127
Autologous iPSC derived RPE cells to treat blindness (md)
- looking at age related macular degeneration which causes RPE cell death
- OKSM reprogramming with episomal vectors from patients skin
- differentiated into RPE in vitro on MEF feeder layers
- cells were screened to check RPE identity and ensure all PSCs gone
- Patient 1 = successfully integrated
- Patient 2 = mutants formed
128
Considerations with iPSC therapies
- must check for left over PSC
- check for mutants
- find alternatives to matrigel and feeder layers
- must use GMP
129
iPSC derived neurones to treat parkinsons in monkeys
- parkinsons = neurodegeneration of dopamine neurones leading to cognitive and motor symptoms
- iPSC from monkey fibroblasts via OKSM RV
- iPSC differentiated into dopamine neurones and transplanted back into monkey
- cells survived and integrated causing a functional recovery
- no tumours and no immunosuppression
130
Liver organoids for transplantation
- use HLA matched iPSC which differentiate into hepatic cells, blood vessel cells and mesenchymal cells
- self assemble into liver bud organoids
- transplant into liver failure mice showed recovery of hepatic function
131
Cholangiocyte organoids as alternative to liver organoids
- donor livers may not have functioning bile ducts
- possibility of injecting cholangiocyte organoids into bile ducts to recover them
- known as ex vivo perfusion models
132
Severe combined immunodeficiency syndrome (CRT RV)
- mutation in the gene coding for IL2RG
- faulty development of T cells and NK cells in the immune system
- Ex vivo RV-IL2RG transduction into an adult immune progenitor cells and inject into patient
- results = persistent proliferation and correction of the disease
- some developed T cell cancer = RV integration?
133
Epidermolysis Bullosa (CRT)
- mutation in LAMB3 prevents normal epidermal anchoring
- RV-LAMB3 into unblistered epidermal cells from patient
- Normal LAMB3 was integrate into the genome of a heterogenous skin cell culture
- Grown into sheet and grafted onto boy
- saw over time all his epidermis was regenerated
134
Mesenchymal stem cell sources
- adult bone marrow
- stromal vascular fraction of adipose tissue
- placenta
- umbilical cord lbood
135
Mesenchymal stem cell characteristics
- heterogenous (problem with GMP)
- wide differentiation potential
- doesnt have a defined marker (problem)
- secretes cytokines and growth factors = paracrine provider
- when injected they home to parts which are inflammed where they will secrete cytokines/GF to stimulate cell repair
- could help with heart attack scar tissue etc
136
Questions to determine if a therapy is legitimate
- efficacy in humans
- safe and effective
- GMP
- kind of cells used
- immunosuppressants
- source of cells
- what is done with extra cells
- does it prevent joining of future clinical trials
- consent
- delivery method
- regulations
- costs
137
eIf2a stress kinases
- HRI
- PKR
- PERK
- GCN2
138
Why HSP can still be made when translation is halted
- HSP undergo cap-independent translation
- the translation that is halted is cap-dependent
139
Ethical issues with HeLa cells
- unjust enrichment
- consent
140
How can HSF1 be activated
- intrinsic heat stress
- HSR1
- Hsp90
Biosci 353
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